The present invention relates to novel polypeptides capable of interacting with human topoisomerase IIIxcex1 and to the nucleic acid sequences encoding these polypeptides. It also relates, in addition, to a method for identifying compounds capable of interacting with said polypeptides and to a method for identifying molecules capable of modulating the interaction of topoisomerase IIIxcex1 with said polypeptides.
The replication of DNA is a complex mechanism which involves a large number of factors. DNA exists in the physiological state in a supercoiled form and access to the information which it contains requires substantial modification of the degree of coiling. Replication requires the suppression of the supercoils, the separation of the two strands of the DNA double helix and the maintaining of DNA in single-stranded form.
The modification of the degree of coiling is brought about in vivo by topoisomerases which are enzymes capable of modifying the DNA superstructures. It is possible to distinguish type I topoisomerases which cut only one of the two DNA strands and which eliminate the supercoils, and type II topoisomerases which act by cutting the two DNA strands and which are capable of eliminating or creating the supercoils. Eukaryotic topoisomerases are less well known than their prokaryotic homologs and their mechanism of action has still not yet been elucidated to date.
The separation of the two strands of a DNA duplex is catalyzed by a group of enzymes, called DNA helicases, which act in an ATP-dependent manner so as to produce the single-stranded DNA used as template for the DNA replication and transcription processes. Generally, the helicases bind to the single-stranded DNA or to the junctions between the single- and double-stranded DNA, and move in a single direction along the DNA in the double-stranded region, destroying the hydrogen bonds joining the two strands. All helicases exhibit a DNA-dependent ATPase (or. NTPase) activity which hydrolyzes the gamma phosphate of the ribonucleoside or deoxyribonucleoside 5xe2x80x2-triphosphate and provides the energy necessary for the reaction. The first helicase was discovered in E. coli in 1976. Since then, more than 60 helicases have been isolated in prokaryotes and eukaryotes. The role of human helicases has still not been elucidated in most cases, with the exception of HDHII (repair of the lesions induced by X-rays), HDHIV (assembly of preribosomes), ERCC2 and ERCC3, which are involved in repair through excision and cell viability. Little is known on the structure of these helicases. A large portion of the information available on the structures and functions of helicases has been obtained by comparative analysis of the amino acid sequences. In particular, conserved motifs have made it possible to group helicases into subfamilies based on the sequence homologies.
Human Topoisomerase III belongs to the family of type IA topoisomerases and therefore exhibits sequence homologies with E. coli topoisomerases I and III, yeast Topoisomerase III as well as reverse gyrase from archaebacteria. Human Topoisomerase III is now called Topoisomerase IIIxcex1 so as to differentiate it from human topoisomerase IIIxcex2 which was recently discovered during the sequencing of the human immunoglobulin xcex gene locus (Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K., Shintani, A., Schmeits, J. L., Wang, J. and Shimizu, N. 1997, Genome Research 7: 250-261), and for which no functional activity has been shown. Yeast-expressed and unpurified topoisomerase IIIxcex1 exhibits an activity of partial relaxation of a highly negatively supercoiled DNA (Hanai, R., Caron, P. R. and Wang, J. C. 1993. Proc. Natl. Acad. Sci. USA 93: 3653-3657).
Topoisomerase IIIxcex1 is a protein of 976 amino acids and with a molecular weight of about 110 kDa. The gene encoding human Topoisomerase IIIxcex1 is present in a single copy on chromosome 17p11.2-12 (Hanai, R., Caron, P. R. and Wang, J. C. 1996. Proc. Natl. Acad. Sci. USA 93: 3653-3657). A murine homolog of Topoisomerase III was recently cloned (Seki, T., Deki, M., Katada, T. and Enomoto, T. 1998. Biochim Biophys Acta 1396: 127-131).
Topoisomerase IIIxcex1 exhibits a strong sequence homology with yeast Topoisomerase III, namely 44% sequence identity and 61% similarity. The homology which it exhibits with bacterial topoisomerases I and III is less strong, namely 24% identity and 44% similarity. However, Topoisomerase IIIxcex1 resembles E. coli Topoisomerase I more than it resembles the other members of the group of type IA topoisomerases from the point of view of the organization of the protein into domains. Indeed, these two polypeptides contain a C-terminal domain which has no equivalent in E. coli or yeast Topoisomerase III. This C-terminal domain contains motifs with 4 cysteines (3 motifs for E. coli Topoisomerase I and 1.5 motif for human Topoisomerase IIIxcex1), as well as an extreme C-terminal domain for which a DNA-binding role has been demonstrated for E. coli Topoisomerase I.
The role of human topoisomerase IIIxcex1 in the cell has not yet been identified.
Human Topoisomerase IIIxcex1 appears to be essential, at least during embryogenesis, since the knock-out of the murine homolog of Topoisomerase IIIxcex1 is lethal (Li, W. and Wang, J. C. 1998 Proc. Natl. Acad. Sci. USA 95: 1010-1013). The messenger RNAs for Topoisomerase IIIxcex1 are present in numerous tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) in the form of three transcripts of 7.2, 6 and 4 kilobases in size (Fritz, E., Elsea, S. H., Patel, P. I. and Meyn, M. S. 1997 Proc. Natl. Acad. Sci. USA 94: 4538-4542).
Moreover, it has been assumed that Topoisomerase IIIxcex1 plays a role in maintaining the stability of the genome. Indeed, the cDNA CAT4.5, encoding a truncated human Topoisomerase IIIxcex1 of 141 N-terminal amino acids, is capable of complementing the phenotype for hypersensitivity to ionizing radiation in AT (Ataxia-Telangectasia) cells exhibiting a mutation in the ATM gene (Fritz, E., Elsea, S. H., Patel, P. I. and Meyn, M. S. 1997 Proc. Natl. Acad. Sci. USA 94: 4538-4542).
In yeast, two independent studies have shown the existence of an interaction between the helicase SGS1 and yeast Topoisomerase III. On the one hand, the sgs1- mutants are suppressers of the top3- phenotype (slow growth, hyperrecombination) in the yeast S. cerevisiae (Gangloff, S., McDonald, J. P., Bendixen, C., Arthur, L. and Rothstein, R. 1994. Mol. Cell. Biol. 14: 8391-8398). On the other hand, it has been shown that the first 500 amino acids of SGS1 interact with yeast Topoisomerase III (Gangloff, S., McDonald, J. P., Bendixen, C., Arthur, L. and Rothstein, R. 1994. Mol. Cell. Biol. 14: 8391-8398, Lu, J., Mullen, J. R., Brill, S. J., Kleff, S., Romeo, A. M. and Sternglanz, R. 1996. Nature 383: 678-679). However, to date, no interaction between a helicase and human Topoisomerase IIIxcex1 has been identified.
The identification of partners of human topoisomerase IIIxcex1 therefore constitutes a major challenge for the understanding of the role of human topoisomerase IIIxcex1, and of its mechanism of action.
The present invention results from the demonstration of novel polypeptides capable of interacting with topoisomerase IIIxcex1 (called hereinafter polypeptide partners of topoisomerase IIIxcex1). It also results from the discovery that these polypeptides show a strong homology with proteins which exhibit structural characteristics common to RNA helicases and for which no function had so far been described. The demonstration of this interaction and of these homologies designate these proteins as DNA helicase partners of topoisomerase IIIxcex1. The identification of these partners makes it possible to envisage numerous applications based on the combined action of these partner proteins and of topoisomerase IIIxcex1; these applications relate in particular to:
1) The destruction of the nucleosomal structure: to undergo some processes such as replication, transcription, repair or recombination, DNA should be accessible to the corresponding enzymatic machineries and, to do this, the nucleosomal structure should be transiently destroyed. It is thus possible to envisage that helicase locally separates the DNA strands and creates positive supercoils ahead of it and negative supercoils behind it. The positive twist is absorbed by the disruption of the nucleosomes, while the negative twist is selectively relaxed by type IA topoisomerase.
2) The positive supercoiling of DNA: the interaction between helicase and type IA topoisomerase is likely to reconstitute in a eukaryotic organism the reverse gyrase activity of thermophilic archaebacteria. Indeed, it has been shown that Sulfolobus acidocaldarius reverse gyrase possesses at the N terminus a helicase domain containing the 8 motifs of helicases with a xe2x80x9cDEADxe2x80x9d motif, and at the C terminus a topoisomerase domain homologous to the type IA topoisomerases (Confalonieri, F., Edie, C., Nadal, M., Bouthier de la Tour, C., Forterre, P. and Duguet, M. 1993. Proc. Natl. Acad. Sci. USA 90: 4753-4757); this enzyme relaxes the negatively supercoiled DNA and introduces positive supercoils into the circular DNA in an ATP-dependent manner (Forterre, P., Mirambeau, G., Jaxel, C., Nadal, M. and Duguet, M. 1985. EMBO J. 4: 2123-2128). This eukaryotic reverse gyrase activity can serve to eliminate particular DNA structures such as the cruciform DNA, the Z DNA, mismatches, recombination intermediates, and the like. From these observations and from the demonstration that topoisomerase IIIxcex1 is capable of interacting with a protein possessing the properties of a DNA helicase, it is possible to envisage the production in vivo or in vitro of a topoisomerase IIIxcex1/protein partner complex constituting an enzymatic complex having reverse gyrase type functions. It should be noted that such a function of positive supercoiling of DNA has still never been described in eukaryotes.
3) The segregation of newly replicated chromosomes: at the end of the replication of DNA, topological problems appear at the level of the point of convergence of two replication forks. A mechanism which makes it possible to solve this topological problem involves the concerted action of a helicase and a type IA topoisomerase, capable of decatenating two single-stranded DNA molecules. This model (Wang, J. C. 1991. J. Biol. Chem. 266: 6659-6662; Rothstein, R. and Gangloff, S. 1995. Genome Research 5: 421-426) proposes that at the point where two replication forks meet, replication is stopped, leaving portions of entangled single-stranded DNAs. These are then separated by means of the concerted action of helicase and topoisomerase. The synthesis of DNA is then completed at the level of the single-stranded regions.
4) The recombination and the stability of the genome: it has been shown that mutants of Top3- yeast or Sgs1- mutants both exhibit a hyperrecombination phenotype while Top3-/Sgs1- double mutants recover a normal phenotype. This shows that yeast Topoisomerase III and helicase SGS1 probably act in a concerted manner to maintain a low rate of recombination, for example by a positive supercoiling activity of the reverse gyrase type, or by a more direct mechanism at the level of the pairings of the recombination intermediates.
Unlike the helicase SGS1, known to interact with yeast topoisomerase III, the protein partner of topoisomerase IIIxcex1 identified by the applicant does not belong to the family of RecQ type helicases.
The polypeptides according to the invention show a high degree of homology with the sequence of a human protein DDX14 published by Chung et al (Chung, J., Lee, S-G., and Song, K. 1995. Korean J. Biochem. 27: 193-197). The protein DDX14 exhibits a significant sequence homology with an RNA helicase of murine origin; however, the helicase activity of this protein has not yet been demonstrated and the function of DDX14 has not yet been elucidated.
The polypeptides according to the invention also show a high degree of homology with the sequence of a human protein DBX1 published by Lahn et al (Lahn, T. and Page, D. C. 1997. Science. 278: 675-680). The protein DBX1 encodes a protein which exhibits homologies with RNA helicases but its helicase activity has never been demonstrated and the function of the DBX1 protein has not yet been identified.
The DBX1 protein encodes a protein of 662 amino acids. The corresponding gene is situated on the X sex chromosome and its homolog situated on the Y chromosome is 91% identical at the protein level. The nucleic and polypeptide sequences of DBX1 are presented in the sequences SEQ ID No. 5 and SEQ ID No. 6. The expression of the DBX1 gene appears to be ubiquitous. It has now been demonstrated that the DBX1 protein possesses the 8 motifs characteristic of helicases of the xe2x80x9cDEADxe2x80x9d family. More precisely, it belongs to the subfamily represented by the helicase PL10, and whose recorded members are the helicases DED1 and DBP1 from yeast, the helicase An3 from amphibians and the murine helicases PL10, mDEAD2 and mDEAD3 (Gee, S. L. and Conboy, J. G. 1994. Gene 140: 171-177). Helicases belonging to this subfamily contain, in addition to the central catalytic domain containing, the 8 conserved motifs of helicases, particular N- and C-terminal domains. The C-terminal domain is rich in arginines and serines, which resembles the domains of splicing factors. However, in the case of the helicases of this subfamily, this domain rich in arginines and serines is shorter and does not possess as many RS dipeptides as in the prototype domain of splicing factors.
The invention also provides a method for identifying molecules capable of blocking the interaction between human Topoisomerase IIIxcex1 and a polypeptide partner of topoisomerase IIIxcex1. Such a method makes it possible to identify molecules which are in particular capable of blocking the reverse gyrase type activity of these two proteins. Such molecules are useful for modulating the processes of division, replication, transcription, translation, splicing, repair or recombination of DNA. These molecules are also capable of possessing a cytotoxic type antitumor activity because of the disruption of these basic processes at the level of the DNA.
A first subject of the invention therefore relates to nucleotide sequences encoding polypeptides capable of interacting with topoisomerase IIIxcex1.
Preferably, the nucleotide sequences according to the invention encode a polypeptide comprising all or part of the polypeptide sequence described in the sequence SEQ ID No. 4 or its derivatives.
For the purposes of the present invention, the term derived polypeptide sequence denotes any polypeptide sequence differing from the sequence considered, obtained by one or more modifications of a genetic and/or chemical nature, and possessing the capacity to interact with topoisomerase IIIxcex1. Modification of a genetic and/or chemical nature is understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated with different aims, such as in particular that of improving its levels of production, that of increasing its resistance to proteases or of improving its passage across the cell membranes, that of increasing its therapeutic efficacy or of reducing its side effects, that of increasing the affinity of the peptide for its site of interaction, or that of conferring novel pharmacokinetic and/or biological properties on it. Advantageously, the variants comprise deletions or mutations affecting amino acids whose presence is not decisive for the activity of the derivative. Such amino acids may be identified for example by tests of cellular activity as described in the examples.
Preferably still, the nucleotide sequences according to the present invention comprise all or part of the nucleotide sequence described in the sequence SEQ ID No. 3 and encoding the sequence SEQ ID No. 4 or the sequences derived from this nucleotide sequence.
For the purposes of the present invention, the term derived nucleotide sequence denotes any sequence differing from the sequence considered because of the degeneracy of the genetic code, obtained by one or more modifications of a genetic and/or chemical nature, as well as any sequence hybridizing with these sequences or fragments thereof and encoding a polypeptide capable of interacting with Topoisomerase IIIxcex1. The expression modification of a genetic and/or chemical nature is understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. The term derivative also comprises the sequences homologous to the sequence considered, which are derived from other cellular sources and in particular from cells of human origin, or from other organisms. Such homologous sequences may be obtained by hybridization experiments. The hybridizations may be carried out starting with nucleic acid libraries, using the native sequence or a fragment thereof as probe, under variable hybridization conditions.
The nucleotide sequences according to the invention may be of artificial origin or otherwise. They may be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semisynthetic sequences. These sequences may be obtained for example by screening DNA libraries (cDNA library, genomic DNA library) by means of probes produced on the basis of sequences presented above. Such libraries may be prepared from cells of different origins by conventional molecular biology techniques known to persons skilled in the art. The nucleotide sequences of the invention may also be prepared by chemical synthesis or by mixed methods including chemical or enzymatic modification of sequences obtained by the screening of libraries. In general, the nucleic acids of the invention may be prepared according to any technique known to persons skilled in the art.
The subject of the present invention is also polypeptides capable of interacting with topoisomerase IIIxcex1.
For the purposes of the present invention, the name topoisomerase IIIxcex1 covers human topoisomerase IIIxcex1 in itself as well as the homologous forms corresponding in particular to mutated forms of this protein.
Preferably, the polypeptides according to the invention comprise all or part of the polypeptide sequence described in SEQ ID No. 4 or of its derivatives.
The present invention also includes a polypeptide characterized in that it is a fragment of the DBX1 protein, capable of interacting with topoisomerase IIIxcex1 and comprising all or part polypeptide fragment which extends between residues 318-662 and represented in the polypeptide sequence SEQ ID No. 6 or its derivatives.
The subject of the present invention is also the use of the polypeptides according to the invention or of fragments of these polypeptides, for slowing down, inhibiting, stimulating or modulating the activity of topoisomerase IIIxcex1.
Indeed, it is possible to envisage regulating the function of topoisomerase IIIxcex1 by means of the polypeptides according to the invention or of fragments thereof and in particular inhibiting or slowing down the activity of topoisomerase IIIxcex1. This modification of the activity of topoisomerase IIIxcex1 is capable of leading to a slowing down of cellular growth or a blocking of the cell cycle or of inducing apoptosis.
Another subject of the present invention relates to a method for preparing the polypeptides according to the invention according to which a cell containing a nucleotide sequence encoding said polypeptides is cultured under conditions for expressing said sequence and the polypeptide produced is recovered. In this case, the part encoding said polypeptide is generally placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, leader sequence for secretion, and the like) may vary according to the cellular host used. Moreover, the nucleotide sequences of the invention may form part of a vector which may be autonomously replicating or integrative. More particularly, autonomously replicating vectors may be prepared using autonomously replicating sequences in the chosen host. As regards integrative vectors, these may be prepared, for example, using sequences homologous to certain regions of the genome of the host, allowing, through homologous recombination, the integration of the vector.
The subject of the present invention is also host cells transformed with a nucleic acid comprising a nucleotide sequence according to the invention. The cellular hosts which can be used for the production of the polypeptides of the invention by the recombinant route are both eukaryotic and prokaryotic hosts. Among the suitable eukaryotic hosts, animal cells, yeasts or fungi may be mentioned. In particular, as regards yeasts, yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula may be mentioned. As regards animal cells, the insect cells Sf9, the cells COS, CHO, C127, of human neuroblastomas, and the like, may be mentioned. Among the fungi, Aspergillus ssp. or Trichoderma spp. may be more particularly mentioned. As prokaryotic hosts, the use of the following bacteria E. coli, Bacillus or Streptomyces is preferred.
According to a preferred mode, the host cells are advantageously represented by recombinant yeast strains.
Preferably, the host cells comprise at least one sequence or one fragment of a sequence chosen from the nucleotide sequences SEQ ID No. 3 or SEQ ID No. 5, for the production of the polypeptides according to the invention.
The nucleotide sequences according to the invention may be incorporated into viral or nonviral vectors, allowing their administration in vitro, in vivo or ex vivo.
Another subject of the invention relates, in addition, to any vector comprising a nucleotide sequence encoding a polypeptide according to the invention. The vector of the invention may be for example a plasmid, a cosmid or any DNA not encapsulated by a virus, a phage, an artificial chromosome, a recombinant virus, and the like. It is preferably a plasmid or a recombinant virus.
As viral vectors in accordance with the invention, there may be most particularly mentioned vectors of the adenovirus, retrovirus, adeno-associated virus, herpesvirus or vaccina virus type. The subject of the present application is also defective recombinant viruses comprising a heterologous nucleic sequence encoding a polypeptide according to the invention.
Another subject of the invention consists in polyclonal or monoclonal antibodies or antibody fragments directed against a polypeptide as defined above. Such antibodies may be generated by methods known to persons skilled in the art. In particular, these antibodies may be prepared by immunizing an animal against a polypeptide whose sequence is chosen from the sequences SEQ ID No. 4 or SEQ ID No. 6 or any fragment or derivative thereof, and then collecting blood and isolating antibodies. These antibodies may also be generated by preparing hybridomas according to techniques known to persons skilled in the art. The antibodies or antibody fragments according to the invention may in particular be used to inhibit and/or reveal the interaction between topoisomerase IIIxcex1 and the polypeptides as defined above.
Another subject of the present invention relates to a method for identifying compounds capable of binding to the polypeptides according to the invention. The identification and/or isolation of these compounds or ligands may be carried out according to the following steps:
a molecule or a mixture containing various molecules, optionally unidentified, is brought into contact with a polypeptide of the invention under conditions allowing the interaction between said polypeptide and said molecule in the case where the latter might possess affinity for said polypeptide, and,
the molecules bound to said polypeptide of the invention are detected and/or isolated.
According to a particular mode, such a method makes it possible to identify molecules capable of blocking the helicase type activity, in particular the DNA helicase activity of the DBX1 protein or of the polypeptides according to the invention and thus modulate the processes of division, replication or transcription of DNA. These molecules are capable of possessing a cytotoxic type antitumor activity because of the disruption of these basic processes at the level of the DNA.
In this regard, another subject of the invention relates to compounds or ligands capable of binding to the polypeptides according to the invention and capable of being obtained according to the method defined above.
Another subject of the invention relates to the use of a compound or of a ligand identified and/or obtained according to the method described above as a medicament. Such compounds are indeed capable of being used for the prevention, improvement or treatment of certain conditions involving a cell cycle dysfunction.
The subject of the invention is also any pharmaceutical composition comprising, as active ingredient, at least one ligand obtained according to the method described above.
Another subject of the present invention relates to a method of identifying compounds capable of modulating or of completely or partially inhibiting the interaction between topoisomerase IIIxcex1 and the polypeptides according to the invention or the DBX1 protein.
The identification and/or isolation of modulators or ligands capable of modulating or of completely or partially inhibiting the interaction between topoisomerase IIIxcex1 and the polypeptides according to the invention or the DBX1 protein may be carried out according to the following steps:
the binding of topoisomerase IIIxcex1 or of a fragment thereof to a polypeptide according to the invention is carried out;
a compound to be tested for its capacity to inhibit the binding between topoisomerase IIIxcex1 and the polypeptides according to the invention is added;
it is determined whether topoisomerase IIIxcex1 or the polypeptides according to the invention are displaced from the binding or prevented from binding;
the compounds which prevent or which impede the binding between topoisomerase IIIxcex1 and the polypeptides according to the invention are detected and/or isolated.
In a particular mode, this method of the invention is suited to the identification and/or isolation of agonists and antagonists of the interaction between topoisomerase IIIxcex1 and the polypeptides of the invention. Still according to a particular mode, the invention provides a method for identifying molecules capable of blocking the interaction between human Topoisomerase IIIxcex1 and the helicase DBX1.
Such a method makes it possible to identify molecules capable of blocking the reverse gyrase type activity of these two proteins and thus modulate the processes of division, replication, transcription, translation, splicing, repair or recombination of DNA. These molecules are capable of possessing a cytotoxic type antitumor activity because of the disruption of these basic processes at the level of the DNA.
In this regard, another subject of the invention relates to compounds or ligands capable of interfering at the level of the interaction between topoisomerase IIIxcex1 and the polypeptides according to the invention or the DBX1 protein and which are capable of being obtained according to the method defined above.
The invention also relates to the use of a compound or of a ligand identified and/or obtained according to the method described above as a medicament. Such compounds are indeed capable of being used for the prevention, improvement or treatment of certain conditions involving a cell cycle dysfunction.
The subject of the invention is also any pharmaceutical composition comprising, as active ingredient, at least one ligand obtained according to the method described above.
Other advantages of the present invention will emerge from reading the examples which follow and which should be considered as illustrative and nonlimiting.